Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase

Abstract

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (D)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

“…Saccharomyces cerevisiae expresses a sugar alcohol phosphatase to prevent the inhibition of glycolysis by sugar alcohol phosphates. The phosphatase hydrolyzes sugar alcohol phosphates into corresponding sugar alcohols, and its expression is positively linked to growth [ 35 ]. The PYP is moderately active against erythrose-4-P, an intermediate in the erythritol biosynthesis pathway suggesting that it could increase erythritol production in Y. lipolytica.…”

“…However, we did not observe increased erythritol expression when TKL was overexpressed. Aside from native genes, we also tested whether the expression of a recently characterized sugar alcohol phosphatase from in S. cerevisiae would increase production [ 35 ]. We found that overexpression of the PYP gene increased erythritol production and glycerol utilization rates in the Y. lipolytica strain.…”

“…In this work, we investigated whether the heterologous expression of a sugar alcohol phosphatase ( PYP ), recently identified in Saccharomyces cerevisiae [ 35 ], would increase sugar alcohol production in Y. lipolytica PO1f, a derivative of Y. lipolytica W29 [ 36 ]. This PYP from S. cerevisiae prevents growth inhibition by sugar alcohol phosphates by hydrolyzing them to produce the corresponding sugar alcohols [ 35 ]. We first tested whether Y. lipolytica natively produces sugar alcohols during growth on different substrates with erythritol being the most promising.…”

Metabolite discovery through global annotation of untargeted metabolomics data

“…Global optimization, integrating all new information comprehensively with prior knowledge to arrive at optimal annotations, is novel and potentially transformative for the field more broadly. The cycle Methods Yeast metabolomics sample preparation and isotope labeling S. cerevisiae strain FY4 was grown for at least 10 generations in minimal essential media containing 0.4% [U- 12 C] or [U- 13 C] glucose and 10 mM ammonium sulfate with or without 0.4 mg/L thiamine hydrochloride 30 . Then, in mid-exponential phase, 5 mL culture broth (OD600 = 0.80) was filtered and metabolites were extracted using 1 mL extraction buffer (40:40:20:0.5 acetonitrile:methanol:water:formic acid), followed by adding 88 μL neutralization buffer (15% NH4HCO3).…”

“…S. cerevisiae strain FY4 was grown for at least 10 generations in minimal essential media containing 0.4% [U- 12 C] or [U- 13 C]glucose and 10 mM ammonium sulfate with or without 0.4 mg/L thiamine hydrochloride 53 . Then, in mid-exponential phase, 5 mL culture broth (OD 600 = 0.80) was filtered and metabolites were extracted using 1 mL extraction buffer (40:40:20:0.5 acetonitrile:methanol:water:formic acid), followed by adding 88 μL neutralization buffer (15% NH 4 HCO 3 ).…”